Venezuelan equine encephalitis virus (VEE) is a member of the alphavirus genus of the Togaviridae. The viral genome is a single-stranded, messenger-sense RNA, modified at the 5'-end with a methylated cap, and at the 3'-end with a variable-length poly (A) tract. Structural subunits containing a single viral protein, C, associate with the RNA genome in an icosahedral nucleocapsid. In the virion, the capsid is surrounded by a lipid envelope covered with a regular array of transmembranal protein spikes, each of which consists of a heterodimeric complex of two glycoproteins, E1 and E2 (Pedersen and Eddy, 1974). Although incompletely studied, the organization of the VEE genome and the overall strategy of VEE gene expression presumably parallel those of the prototype alphaviruses, Sindbis virus and Semliki Forest virus (reviewed in Schlesinger and Schlesinger, 1986). For example, details of the partial genome sequence (Kinney et al., 1986) demonstrate that VEE structural proteins are translated in the form of a polyprotein from a 26S subgenomic mRNA which corresponds to the 3'-one third of the viral genome. Proteolytic processing produces the proteins found in the mature virion. Alphavirus nonstructural protein genes are located in the 5'-two thirds of the genome in the order nsP1, nsP2, nsP3 and nsP4. The proteins are expressed initially as polyprotein pre cursors and then proteolytically processed to their mature forms (Keranen and Ruohonen, 1983; Strauss et al., 1984; Hardy and Strauss, 1988). The mature nonstructural proteins are required or replication of genome RNA and the synthesis of 26S subgenomic mRNA.
VEE, which was first isolated during a serious epizootic in Venezuela in the mid 1930's (Kubes and Rios, 1939), continues to be a significant public health problem in South and Central America. The natural enzootic cycle has been described in many areas, reaching as far north as Florida. In these foci, the virus can be isolated from mosquitoes as well as equines and other vertebrate species. The equine disease induced by enzootic strains is relatively benign (Henderson et al., 1971; Scherer and Chin, 1977). In contrast, combined data from the study of VEE epizootics show involvement of different mosquito species and antigenically distinct strains of VEE (Young and Johnson, 1969; Scherer and Chin, 1977; Trent et al., 1979). Epizootic strains of VEE cause much more severe equine disease with fatality rates as high as 83% (Groot, 1972). Acute febrile disease in humans is associated with VEE epidemics, is often very widespread, but is usually relatively mild. Severe neurologic disease, including fatal encephalitis, is most frequent in children, with case-fatality rates as high as 0.7% (Groot, 1972).
Preventive measures include yearly vaccination of horses with an inactivated VEE vaccine and control of mosquito populations. An experimental live vaccine, TC-83 (Berge et al., 1961) was used to protect large numbers of horses during the 1971 Texas outbreak, and is routinely used to immunize laboratory workers. The serious side effects and less than 100% effectiveness associated with this vaccine limit its usefulness for humans. The unpredictable nature of VEE epidemics, the demonstrated capability of VEE to spread over large areas, the increased virulence of epidemic strains, and the inadequacies of the TC-83 vaccine indicate a need for further understanding of VEE epidemiology, biology and pathogenesis.
Full-length cDNA clones of positive-strand RNA viruses are important tools for the study of the biology of this group of viruses. It has been demonstrated in numerous virus systems that in vitro transcripts of cDNA clones, and in some cases the cDNA itself, can initiate a complete and productive infectious cycle upon introduction into susceptible cells (for examples, see Racaniello and Baltimore, 1981; Ahlquist et al., 1984; Kaplan et al., 1985; Mizutani and Colonno, 1985; van der Werf, 1986; Rice et al., 1987; Vos et al., 1988). This has made it possible to test progeny virus for phenotypic manifestations of directed mutations and recombinations which have been introduced into the cDNA clone. Pathogenesis studies with several positive-strand viruses, including the picornaviruses (La Monica et al., 1986; La Monica et al., 1987; Nomoto et al., 1987) and the alphaviruses (Polo et al., 1988; Lustig et al., 1988) have been advanced significantly by the use of full-length cDNA clones.
We herein describe the construction of a cDNA clone of the VEE genome downstream from a synthetic T7 promoter. In vitro transcription of this clone with the T7 RNA polymerase yielded infectious VEE RNA. In addition, analysis of this cDNA clone led to the discovery of a large sequence duplication near the 3'-end of the VEE nsP3 gene.